Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983, 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error prone process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage. Clinical trials using cells modified using engineered nucleases have demonstrated therapeutic utility (see, e.g. Tebas et al (2014) New Eng J Med 370(10):901).
Targeted cleavage using one of the above mentioned nuclease systems can be exploited to insert a nucleic acid into a specific target location using either HDR or NHEJ-mediated processes. However, delivering both the nuclease system and the donor to the cell can be problematic. For example, delivery of a donor or a nuclease via transduction of a plasmid into the cell can be toxic to the recipient cell, especially to a cell which is a primary cell and so not as robust as a cell from a cell line. Plasmid DNA contains several elements required for its production in bacteria, and is subject to modifications that mark the plasmid as foreign to mammalian cells. Therefore, transfection or nucleofection of plasmid DNA into human cells can cause toxicity. Indeed, genome engineering and transgenic insertion is often an inefficient process due at least in part to the toxicity of the DNA constructs.
DNA minicircles (MCs) are supercoiled DNA molecules that can be used for non-viral gene transfer that have neither an origin of replication or a antibiotic selection marker. These DNAs are devoid of bacterial DNA, and thus lack the unmethylated CpG motifs found in bacterial DNA. These CpG motifs have been shown to active the innate immune response in mammals by binding to the Toll-like receptor 9 receptors on antigen presenting cells. Thus, use of DNAs for gene therapy that contain bacterially derived DNA sequence may be more inflammatory that those DNAs lacking bacterial sequences. MCs are smaller than standard plasmids used in some gene therapy applications, and are more efficiently transfected into both cell lines than standard plasmids (Darquet et al (1999) Gene Therapy 6:209-218) and T cells (Sharma et al (2013) Molecular Therapy-Nucleic Acids 2 e74). Additionally, DNA MCs are useful for plant cell transformation by either direct DNA uptake by a plant cell, or by use of standard techniques such as Agrobacterium mediated transformation or passive uptake; the use of electroporation; treatments with polyethylene glycol; electrophoresis; cell fusion with liposomes or spheroplasts; microinjection, silicon carbide whiskers, and particle bombardment (U.S. Patent publication 20120042409). The MCs can be made through the exploitation of phage integrase φC31-mediated site specific recombination between the attB and attF sites (see Darquet, ibid) and can be produced at large scale.
Thus, there remains a need for compositions and methods for delivery of nucleic acids needed for nuclease-mediated genome engineering to cells that are less toxic and more efficient than currently available methods.